The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin.

The production of five monoclonal antibodies to the epsilon prototoxin of Clostridium perfringens is described. All five monoclonal antibodies located three proteins in a trypsinized preparation of epsilon prototoxin. These proteins were located at 37.6 kDal, 35.6 kDal and 33.7 kDal by Western blots. Two of the monoclonal antibodies, M26/2 and M27/12, neutralized epsilon toxin in the mouse lethality assay. Four of the five monoclonal antibodies are considered suitable as reagents to detect epsilon toxin in assay procedures.

Production and evaluation of monoclonal antibodies directed against the K88 fimbrial adhesin produced by Escherichia coli enterotoxigenic for piglets.

Fifty murine monoclonal antibodies were produced against a purified preparation of K88ab antigen. Two monoclonal antibodies were selected for further studies together with a monoclonal antibody directed against the c region of K88 (5CA3). Monoclonal antibody K88-13 bound to all K88+ Escherichia coli examined, whereas K88-18 and 5CA3 bound to K88ab+ and K88ac+ strains, respectively. The monoclonal antibodies failed to react with K88+ E coli grown at 18 degrees C or with K88- E coli grown at 37 degrees C or 18 degrees C. Binding of monoclonal antibody K88-13 to K88 antigen was blocked by OK antisera to G7 (O8:K87 K88ab) and Abbotstown (O149:K91 K88ac), whereas absorbed antisera to K88b, c and d had no effect. Monoclonal K88-18 was inhibited by OK G7 antiserum and absorbed antiserum to K88b. One hundred and forty-nine strains of K88+ E coli representing seven somatic O groups were agglutinated by antibody K88-13; 142 of these, from at least six somatic O groups were agglutinated by 5CA3 and the remainder by K88-18. Monoclonal antibody K88-13 bound to cryostat sections of ileum from piglets infected with E coli strains Abbotstown and G7. K88-18 and 5CA3 bound only to sections from piglets infected with G7 and Abbotstown strains, respectively. It is concluded that monoclonal antibody K88-13 recognises an epitope on the a region of K88 while monoclonal antibody K88-18 is directed towards the b region. These monoclonal antibodies together with 5CA3 can be used to routinely identify K88+ E coli isolates.

Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus ticks.

A rapid method is described for preparing and staining salivary glands of Rhipicephalus appendiculatus ticks infected with Theileria parva. The technique, involving the use of a modified methyl green pyronin stained minimizes the risk of losing material and allows examination of stained glands within minutes of preparation. The technique was applied in a series of studies in which ticks were either infected with T. parva under different conditions, or maturation of parasites in adult ticks was stimulated by different means. When nymphal ticks were fed on the ears of cattle the subsequent infection rate of the adult ticks showed no correlation with the parasitaemia of the cattle at the time of nymphal engorgement. There was no difference in infection rates between adult ticks in which parasite maturation had been stimulated either by incubation at 37 degree C or by feeding on rabbits. However, parasite maturation took about 1 day longer in incubated ticks than in rabbit-fed ticks. Female ticks were consistently more highly infected than males, both in terms of the percentage of ticks infected and the mean number of infected acini/tick. Ticks were infected with T. parva by injection of nymphs with parasitaemic bovine blood, but the resultant adult infection was lower than that in ticks which had been infected naturally by feeding on cattle.

Plasma hormone levels and reproductive behaviour in anoestrous ewes after treatment with progesterone and PMSG.

Plasma progesterone and gonadotrophin levels were studied in anoestrous ewes treated during June or July with a subcutaneous progesterone implant and/or an injection of oestradiol or PMSG. Of 32 ewes treated with progesterone during July, 9 showed a gonadotrophin surge after removal of the implant, and 10 ewes showed oestrous behaviour during the following 4 days. Six ewes conceived at this induced oestrous. Progesterone treatment during June was much less effective, with only 2 of 19 treated ewes showing a gonadotrophin surg and oestrous behaviour. Administration of PMSG at the time of implant removal in the June experiment was followed by a gonadotrophin surge and oestrous behaviour in 18 of 19 ewes, and 15 ewes conceived at the induced oestrus. An injection of PMSG, without progesterone pretreatment, stimulated a gonadotrophin surge and ovulation, but did not result in oestrous behaviour. The treatments employed appeared to initiate cyclic ovarian activity in the July experiment, but not in the June experiment.

The acute effects of oestradiol-17beta and synthetic LH-RH on plasma LH levels in freemartin heifers.

Injection of oestradiol was followed by a surge of plasma LH within 24 h in only 7 of 12 freemartins. Elevations of plasma LH were less than those reported for normal non-cyclic heifers, but some freemartins showed a delayed, or more prolonged, LH response. Responsiveness to oestradiol was not related to degree of chimaerism or plasma androstenedione level, and most of the animals responded similarly in two trials carried out 4 months apart, during which time plasma androstenedione levels had more than doubled. Freemartins which showed an LH surge after oestradiol treatment released greater amounts of LH after the injection of LH-RH than did non-responders.

The acute effect of oestrogen injection on plasma LH in freemartin heifers.

The acute effects on plasma LH concentrations of an injection of oestradiol-17beta were studied in 7 non-cyclic heifers and 19 freemartins. One freemartin showed a normal LH surge due to the positive feedback effect of oestrogen on the hypothalamus. Of the other 18 freemartins, 4 showed positive increases in plasma LH and 6 were unclassified. There was no correlation between the degree of chimaerism and responsiveness to oestrogen. The results also showed that injected oestradiol suppressed the spontaneous fluctuations of plasma LH.

In-vitro studies on intestinal malabsorption of amino-acids in young chicks infected with Eimeria acervulina.

In vitro uptake of L. histidine by intestinal tissue from chicks infected with E. acervulina was slightly reduced. This was confirmed using radio-actively labelled mixed amino-acid substrates in the form of a Chlorella protein hydrolysate.

Amino-acid malabsorption and intestinal leakage of plasma-proteins in young chicks infected with Eimeria acervulina.

The in vivo absorption of 14C-labelled amino-acids and the leakage of radioactive plasma proteins into the gut of infected birds were measured together with their food-intake and growth. The results suggested that anorexia and protein leakage from the gut are major factors in the pathogenesis of E. acervulina infection.

Estimation of the growth of the T 1 strain of Mycoplasma mycoides in tryptose broth by the measurement of lactate dehydrogenase. I. Method.

A highly significant correlation coefficient (r = 0.97, n = 18) was found between the concentration of lactate dehydrogenase measurable after the organisms had been disrupted and the concentration of colony-forming units during the logarithmic phase of growth of a broth culture of the T(1) strain of Mycoplasma mycoides var. mycoides. A concentration of 4.60 x 10(-7) milliunits of lactate dehydrogenase for each colony-forming unit was established. This relationship was used to convert the concentration of lactate dehydrogenase in the culture into an estimate of the concentration of viable mycoplasma. The lactate dehydrogenase was estimated by following the oxidation of reduced nicotinamide adenine dinucleotide, in the presence of pyruvate substrate, at 366 nm in a spectrophotometer. The nicotinamide adenine dinucleotide oxidase system probably contributed a small amount of enzyme activity to the test when lactate dehydrogenase was measured in this way. The method has been described and evaluated for the estimation of titers from 10(7) to 5 x 10(9) colony-forming units per ml.

Estimation of the growth of the T 1 strain of Mycoplasma mycoides in tryptose broth by the measurement of lactate dehydrogenase. II. Application to vaccine production.

The measurement of lactate dehydrogenase activity has been used to evaluate the growth of the T(1) vaccine strain of Mycoplasma mycoides var. mycoides in broth during the production of vaccine. Estimations were made during incubation at 37 C and storage at 4 C. The results were compared with titers of the vaccine obtained by two conventional methods of assessing viability titers: the counting of colonies formed on solid medium and a 50% end point titer found after dilutions were made in broth. The method of measuring lactate dehydrogenase activity allowed the rapid enumeration of organisms during incubation of the culture, but estimations made on vaccine stored at 4 C did not compare well with the two conventional methods of assessing viability. The lactate dehydrogenase activity of the culture has enabled rapid monitoring of the production stages of this vaccine.